Although many analogs of vitamin D metabolites have been synthesized and evaluated for their biological activity, very little is known about the mechanisms responsible for the observed structure-activity relations for these compounds. We have begun a research program that will critically analyze the important structural features of the vitamin D3 molecule that are required for its metabolism and biological activity. As a model we have synthesized 3-epivitamin D3, the 3-epimer of vitamin D, and examined in detail how a small perturbation in the molecular structure of a vitamin D3 analog alters its biological activity. 3-epivitamin D3 is 20% as active as vitamin D3 in stimulating intestinal calcium transport and bone calcium mobilization, and in healing rickets in rats. To understand the reason(s) for this reduced activity, we radiolabeled this analog and compared its metabolism with that of vitamin D3 in vivo and in vitro. 0.25 micrograms of (3 beta-3H)-vitamin D3 were administered to vitamin D-deficient rats. Blood, intestine, liver, kidney, urine and feces were collected at 24 and 48 hours, and lipid extracts were prepared and chromatographed. Like vitamin D3, 3-epivitamin D3 metabolized to two polar compounds. The first peak migrates similarly to 25-OH-D3; in addition, rat liver homogenates metabolize 3-epivitamin D3 to a compound believed to be 25-OH-3-epivitamin D3 which co-elutes with the first peak. The more polar 3-epi-metabolite, the production of which can be blocked by nephrectomy, co-elutes with synthetic 1 alpha, 25-(OH)2-3-epivitamin D3. An analysis of the metabolism of 3-epivitamin D3 demonstrates that 3-epivitamin D3 is converted to 1 alpha, 25-(OH)2-3-epivitamin D3 as efficiently as vitamin D3 is converted to 1 alpha, 25-(OH)2-D3. Thus the decreased biological activity of the analog cannot be explained by inefficient metabolism. An analysis of the target tissue concentration of 1 alpha, 25-(OH)2-3-epivitamin D3 and the intestinal cytosol receptor affinity for 1 alpha, 25-(OH)2-3-epivitamin D3 compared to 1 alpha 25-(OH)2-D3 provides new insights into the importance of the specificity of the cytosol receptor on the intestinal calcium transport response.